Interleukin (IL)-12 is a newly described cytokine that has strongly enhanced cell-mediated immunity through induction of interferon-gamma and activation of cytotoxic T cells and NK cells. However, its effects on B cells and humoral immunity are only poorly understood. We recently demonstrated by flow cytometry the presence of an IL-12 receptor on activated splenic B cells and on normal B1 cells, the B cell subset that is thought to be primarily responsible for autoantibody production and chronic lymphocytic leukemia (CLL). Furthermore, we have shown that in vivo or in vitro exposure to IL-12 stimulates conventional B cells but inhibits the function of B1 cells. Since these effects are not mediated by interferon-gamma, we believe that B cells are directly influenced by IL-12 binding. We now propose to determine the mechanism(s) for the differential effects of IL-12 on B cell subset activity and the applications of IL-12 for modification of B cell function. The influence of cytokine treatment on antibody specificities in T-dependent responses will be determined using hen eggwhite lysozyme and naturally-occurring lysozyme variants that contain known amino acid substitutions. The ability of IL-12 to serve as an adjuvant for T-independent type 1 and type 2 antibody responses will be assessed using N. meningitidis capsular polysaccharide and S. pneumoniae phosphorylcholine (PC), respectively, as model antigens. Potential effects on B1 cells in the anti-PC response and in autoimmune-prone NZBWF(1) mice will be investigated by measuring T15 idiotype production, analyzing cell subset expression by flow cytometry, and monitoring development of autoimmunity. A recently developed in vitro model for the ability of IL-12 to alter Ig isotype production will be utilized to determine whether IL-12 induces heavy chain isotype switching and if it acts directly on B cells or through intermediary cells and/or cytokines. Possible differences in expression of the IL-12 receptor complex between B cell subsets will be assessed by equilibrium binding analyses and immunoprecipitation studies, as well as by differences in JAK-STAT signal transduction pathways. The potential presence of a homologous IL-12 receptor on human B cells will be tested by analysis of human umbilical cord blood and peripheral blood of CLL patients, both rich sources of B1 cells. The ability of IL-12 to influence the activity of human B cell subpopulations will be tested using in vitro and adoptive transfer systems. These experiments will provide important new information about the effects of IL-12 on B lymphocyte function and could ultimately lead to new approaches for the biological modification of human diseases.